FIG. 3.

The HOX-PBX complex associates with class I HDACs in vivo and represses transcription in a mSIN3B/N-CoR/SMRT-dependent manner. (A) PBX1 coprecipitates with class I HDACs (HDAC1 and HDAC3, lane 2 and 4) but not with HDAC4 (lane 3) or from cells transfected with the empty flag vector (F-control) (lane 1). Immunoprecipitations were done with lysates from 293 T cells cotransfected with plasmids expressing PBX1A and flag-tagged HDAC1 (F-HDAC1), F-HDAC3, F-HDAC4, or F-control. Flag-tagged proteins were immunoprecipitated with M2 beads (Sigma), and the precipitates were eluted with flag peptides (Sigma) and analyzed by Western blotting using rabbit polyclonal antibodies against PBX1 (Santa Cruz). “IP” and “WCE” denote immunoprecipitates and whole-cell extracts used in Western blot analysis. “W” denotes the antibody used in Western analysis. (B) Coprecipitation of endogenous HDAC1 and mSIN3B (but not mSIN3A) with rabbit polyclonal antibodies against PBX1. 293 T cells were transfected with a plasmid expressing PBX1A but not with plasmids expressing HDAC1, mSIN3B, or mSIN3A. Immunoprecipitates with anti-PBX1 antibodies (IP: α-PBX1) were analyzed in Western blots with antibodies against HDAC1 (W:α-HDAC1), mSIN3a (W:α-mSIN3a), and mSIN3b (W:α-mSIN3b). (C) The repression of pML(5xHOX-PBX) in 293 T cells is exerted by N-CoR/SMRT-corepressor complexes. Overexpression of either N-CoR or SMRT further repressed pML(5xHOX-PBX). This repression can be partially relieved by sequestering the endogenous N-CoR/SMRT with overexpressed estrogen receptor (ER) bound to the estrogen antagonist TOT (see Materials and Methods). (D) Immunoprecipitation of PBX1 from cells expressing flag-tagged N-CoR (F-N-CoR, lane 2) but not from cells transfected with the empty flag vector (F-control, lane 1).